Molecular parameters characterizing the interaction of Escherichia coli lac repressor with non-operator DNA and inducer

Ap, Butler; Revzin, Arnold; Hippel, Peter H. von

doi:10.1021/bi00641a001

Cited by 95 publications

(75 citation statements)

References 51 publications

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“…The information in this paper was first used to determine t values for proteins by groups at the University of Oregon (Butler et al, 1977;Elwell & Schellman, 1977), and has since become known as the Edelhoch method. A clear description of the method was given by Gill and von Hippel (1989).…”

Section: Edelhoch Methodsmentioning

confidence: 99%

How to measure and predict the molar absorption coefficient of a protein

et al. 1995

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The molar absorption coefficient, E, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring E for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-3261 and is based on data from Edelhoch [ , Biochemistry 6:1948.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average E values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the E values in water, propanol, These ~(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict E .

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Section: Edelhoch Methodsmentioning

confidence: 99%

How to measure and predict the molar absorption coefficient of a protein

et al. 1995

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“…The protein was Ͼ95% pure as judged by SDS-PAGE and Ͼ50% active in lac operator binding (27). Repressor concentrations were determined spectrophotometrically using ⑀ 280 ϭ 2.2 ϫ 10 4 M Ϫ1 cm Ϫ1 /repressor subunit (28). Protein concentrations given throughout this paper refer to the species active in sequence-specific DNA binding.…”

Section: -26)mentioning

confidence: 99%

Role of Hydration in the Binding of lac Repressor to DNA

Fried¹,

Stickle²,

Smirnakis³

et al. 2002

Journal of Biological Chemistry

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The osmotic stress technique was used to measure changes in macromolecular hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260 ؎ 32 water molecules. If all are released from macromolecular surfaces, this result is consistent with a net reduction of solvent-accessible surface area of 2370 ؎ 550 Å 2 . This area is only slightly smaller than the macromolecular interface calculated for a crystalline repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the symmetrical optimized O sym operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93 ؎ 10 water molecules. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165 ؎ 43 water molecules. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concentrations.The control of transcription initiation involves the binding of gene regulatory proteins to regulatory and competing genomic DNA sequences. The stability and specificity of these interactions depend on their solution environment. Important variables include salt concentration and identity (1-3), pH (4, 5), pressure (6 -8), and accessible volume (9, 10). In addition, changes in hydration accompany macromolecular interactions (reviewed in Refs. 8, 11, and 12). For those interactions in which the hydration change is large, the free energies of interaction depend sensitively on the activity of water (a H 2 O ).The intimate association of protein and DNA surfaces is accompanied by the displacement of water molecules associated with those surfaces. In addition, allosteric changes that extend beyond the contact surfaces can alter the solvent-accessible surface areas of protein and DNA and, thus, the numbers of associated water molecules. Any water molecules bound or released in these transactions are reactants or products, respectively, in the binding reaction. Changes in the number of thermodynamically associated water molecules can be detected and quantitated by the osmotic stress technique (12-14), using small, neutral solutes (osmolytes) that are typically excluded from the volumes immediately adjacent to macromolecular surfaces (11, 15). With three caveats, the dependence of the affinity of protein for DNA (K obs ) on water activity is a measure of the net change in the number of water molecules that are associated with the participating macromolecules. These are as follows: (i) that K obs should not be significantly perturbed by the differential interaction of osmolytes with reactants and products; (ii) that volume exclusion by osmolytes should not significantly alter K obs ; and (iii) that changes in solvent properties that indirectly affect bin...

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“…Some DNA-binding proteins exhibit a low to moderate affinity for DNA in general, but a high affinity for certain specific DNA sequences [23,24] ; however, no sequence-specific, DNA-binding sites have yet been demonstrated for steroid hormone receptors [2] . The present study, in fact, is part of a systematic search for possible specificity determinants which may be operative in the binding of these receptors to chromatin.…”

Section: Resultsmentioning

confidence: 99%